The Quantikine Human PTX3 Immunoassay is a 5.0 hour solid-phase ELISA designed to measure human PTX3 in pretreated cell culture supernates, plasma, and saliva. It contains NS0-expressed recombinant human PTX3 and has been shown to accurately quantitate the recombinant factor. Results obtained using pretreated natural human PTX3 showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for naturally occurring human PTX3.

Precision

Intra-Assay Precision (Precision within an assay) Three samples of known concentration were tested on one plate to assess intra-assay precision

Inter-Assay Precision (Precision between assays) Three samples of known concentration were tested in separate assays to assess inter-assay precision

Serum-free Cell Culture Supernates, EDTA Plasma, Saliva

Intra-Assay Precision

Inter-Assay Precision

Sample

1

2

3

1

2

3

n

20

20

20

40

40

40

Mean

2.61

7.72

14.1

2.75

7.94

14.5

Standard Deviation

0.01

0.28

0.62

0.17

0.39

0.6

CV%

3.8

3.7

4.4

6.1

5

4.1

Recovery

The recovery of PTX3 spiked to levels throughout the range of the assay was evaluated.

Sample Type

Average % Recovery

Range %

EDTA Plasma (n=4)

99

83-106

Serum-free Cell Culture Media (n=4)

101

95-108

Linearity

To assess the linearity of the assay, pretreated samples containing and/or spiked with high concentrations of PTX3 were serially diluted with the Calibrator Diluent to produce samples with values within the dynamic range of the assay.

Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: Pentraxin 3/TSG-14

Pentraxin 3 (PTX3), also known as TNF-stimulated gene 14 (TSG-14), is a secreted glycoprotein belonging to the Pentraxin family of proteins. It is the prototypical long Pentraxin, exhibiting a C-terminal Pentraxin domain characteristic of the family, and a unique N-terminal domain. It is produced by several cell types including endothelial and mononuclear cells. PTX3 acts as a pattern recognition receptor with non-redundant roles in the innate immune response to several microbial agents including the fungal pathogen Aspergillus fumigatus. Overexpression leads to enhanced pro-inflammatory responses. PTX3 is induced in response to LPS and inflammatory cytokines including TNF-alpha and IL-1 beta.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

Prepare all reagents, standard dilutions, and samples as directed in the product insert.

Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

100 µL Assay Diluent

Add 100 µL of Assay Diluent to each well.

20 µL Pretreated Standard, Control, or Sample

Add 20 µL of pretreated Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours on a horizontal orbital microplate shaker.

Aspirate each well and wash, repeating the process 3 times for a total of 4 washes.

200 µL Conjugate

Add 200 µL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours on the shaker.

Aspirate and wash 4 times.

200 µL Substrate Solution

Add 200 µL Substrate Solution to each well. Incubate at room temperature for 30 minutes on the benchtop. PROTECT FROM LIGHT.

50 µL Stop Solution

Add 50 µL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.

Citations:

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